Diagnosis of acute disease is often based on immunoassay, e.g. enzyme-linked immunosorbent assay (“ELISA”), detection of abnormal levels of clinical markers, such as proteins, enzymes and hormones in biological fluids, particularly when the concentration changes quickly during the acute phase of disease. ELISA systems allowing for the rapid and simple diagnosis of the occurrence of an acute disease, such as the occurrence of an MI, are therefore extremely important.
In the past, clinical markers for diagnosing the occurrence of an MI included lactate dehydrogenase (“LDH”) and glutamic oxaloacetic transaminase (“GOT”), though these were not very specific. Problems with LDH and GDH led to the use of the MB isoenzyme of creatine kinase (“CK-MB”) as a clinical marker for the diagnosis of MI. However, CK-MB can also be found in skeletal muscle and in blood after skeletal muscle injury. Thus, CK-MB is not completely specific for cardiac muscle. Another disadvantage of CK-MB as a clinical marker of MI is that the level of CK-MB in the skeletal muscle varies with the degree of skeletal muscle regeneration, information which may often not be known when administering a test or analyzing a test result for MI. Another disadvantage of testing for CK-MB is that CK-MB levels remain elevated for only 2-3 days after the onset of chest pain. For patients admitted after that time, the CK-MB test will be of limited value. Thus, due to the lack of specificity when assaying CK-MB, and the limited time frame for its use as a diagnostic tool, CK-MB is not an ideal clinical marker for diagnosing MI.
Cardiac troponin 1 (“cTnI”) is now used as an accurate cardiac-specific biological parameter detectable in serum very soon after MI and remaining present for more than 2-3 days after the onset of MI. Troponin is present in cardiac tissue as a complex of three subunits: Troponin T (“TnT”), the tropomyosin binding subunit, Troponin C (“TnC”), the Ca2+ binding subunit; and Troponin I (“TnI”), the sub-unit which inhibits the actomyosin Mg2+ ATPase. TnI is a thin filament regulatory protein complex, which confers calcium sensitivity to the cardiac and striated muscle.
Human Troponin I exists in three isoforms: two skeletal muscle isoforms (fast and slow) (MW=19.8 kDa) and a cardiac TnI isoform (“cTnI”) with an additional 31 residues on the N-terminus resulting in a molecular weight of 23 kDa (209 amino acids). Cardiac TnI is uniquely located in the myocardium where it is the only isoform present. Cardiac TnI rapidly appears in human serum (within approximately 4 to 6 hours) following a MI. It reaches a peak level after 18-24 hours and remains at elevated levels in the blood stream for up to 6-10 days. As a result, cTnI released into the circulation from the myocardium is very specific for myocardial injury.
Elevated cTnI levels in blood may be used to diagnose MI and distinguish other heart related events and diseases. Immunoassay systems capable of accurately detecting human cTnI would be valuable to the medical community for diagnosing the occurrence of MI. For more information on the utility of cTnI testing, see Apple and Wu, Myocardial infarction redefined: Role of cardiac troponin testing. Clinical Chemistry 47, 377-9, (2001), which is hereby incorporated by reference.
Cardiac TnI exists in multiple subforms in blood as a result of modifications such as proteolytic cleavage, phosphorylation, chemical oxidation, chemical reduction, cleavage of amino acid residues, and chemical modification of amino acid moieties. For example, amino acids 1 to 25 and 150 to 209 are generally not found on cTnI subforms in serum as a result of proteolysis. The many different subforms of cTnI circulating in the bloodstream are predominantly found in complexes with other proteins. See Katrukha (1997). For example, cTnI may be complexed with cTnT and cTnC (“cITC”).
In many instances, the chemical modifications and complexation of cTnI in the bloodstream eliminate or block the binding sites for ELISA reagents on some subforms of cTnI, thereby making the epitopes of the ELISA reagents unavailable for binding. For more information on cTnI epitopes and cTnI instability, see Morjana et al., Degradation of Human cardiac troponin I after myocardial infarction: Biotechnol. Appl. Biochem. (1998), 28, 105-111; Gaelle Ferrieres et al. Human cardiac troponin 1: precise identification of antigenic epitopes and prediction of secondary structure: Clin. Chem. (1998), 44, 487-493; Katrukha et al., Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex: Clin. Chem. (1997), 43, 1379-1385; and Katrukha et al., Degradation of cardiac troponin I: implication for reliable immunodetection: Clin. Chem. (1998), 44, 2433-244, which are incorporated herein by reference.
For most commercially important clinical marker analytes, a number of efficient monoclonal antibodies have been developed which bind a particular epitope of an analyte. The difference between antibodies are the specific location of binding to the analyte, affinity for the analyte, and cross-reactivity with other potential interferents within the sample. ELISA assays have been traditionally developed by pairwise testing of available antibodies for use either as the capture reagent or signal reagent.
In the development of ELISA assays for cTnI, much effort has been expended in optimizing the antibody and enzyme reagents used. However, the likelihood of epitopes being unavailable for binding on the many subforms of cTnI present in serum and complexation with other proteins makes detection of cTnI significantly difficult and typically cause current ELISA reagents to underestimate actual cTnI levels in a sample. Furthermore, the many different subforms of cTnI with different epitopes available for binding cause current ELISA assays to give significantly different results from sample to sample, from reagent set to reagent set, and as a function of time.
Thus, there exists a need to develop methods of detecting a clinical marker and subforms thereof, in particular cTnI, for the diagnosis of an acute disease event, in particular MI. The present invention satisfies this need and provides reagents, kits, and apparatuses for detecting a clinical marker and subforms thereof, in particular cTnI.